In normal tissues, cellular connective tissue synthesis is offset by extracellular matrix degradation, the two opposing effects existing in dynamic equilibrium. Degradation of the matrix is brought about by the action of matrix metalloproteinases (MMPs) released from resident connective tissue cells and invading inflammatory cells. Normally, these catabolic enzymes are tightly regulated at the level of their synthesis and secretion and also at the level of their extracellular activity. Extracellular control occurs primarily by regulation with specific enzymes, such as TIMPs (tissue inhibitors of metalloproteinases), which form complexes with MMPs. These complexes prevent MMP action. Cellular level control of MMP activity occurs primarily by regulating MMP gene expression and down regulating the expression of the membrane bound MMPs (MT-MMP) that activate the excreted proenzyme form of the MMP.
MMPs are a family of neutral metalloenzymes capable of degrading extracellualr matrix (ECM) macromolecules. Members of this family that have been isolated and characterized include interstitial fibroblast collagenase, stromelysin, and type IV collagenase. Other potential members include a poorly characterized 94,000 dalton gelatinase and several low molecular weight gelatinases and telopeptidases. Structurally, MMPs contain a zinc(II) ionic site at the active site of the protein. Binding of zinc to the ionic site is required for hydrolytic activity.
TIMPs are glycoproteins that specifically regulate interstitial collagenase on a 1:1 stoichiometric basis. That is, TIMPs form very specific regulatory complexes with MMPs, only regulating a specific subset of the MMPs. No naturally occurring TIMP molecule singly regulates all types of MMPs.
In chronic wounds, the ratio of MMPs to TIMPs is high, such that most of the MMPs are unregulated. This unregulated MMP activity results in the accelerated, uncontrolled breakdown of the ECM, leading to destruction of the newly formed wound bed. Additionally, the concomitant elevation of proteinase levels, cause hydrolyzation of TIMP molecules, further increasing the MMP to TIMP ratio.
Many individuals suffer from chronic wounds. Open cutaneous wounds represent one major category of such wounds and include burn wounds, neuropathic ulcers, pressure sores, venous stasis ulcers, and diabetic ulcers. Worldwide, eight million people have chronic leg ulcers and seven million people have pressure sores (Clinica 559, 14–17, 1993). In the U.S. alone, the prevalence of skin ulcers is 4.5 million, including two million pressure sore patients, 900,000 venous ulcer patients and 1.6 million diabetic ulcer patients (Med Pro Month, June 1992, 91–94). The cost involved in treating these wounds is staggering and, at an average of $3,000 per patient, reaches over $13 billion per year for the U.S. alone.
Burn wounds have a reported incidence of 7.8 million cases per year worldwide, 0.8 million of which need hospitalization (Clinica 559). In the U.S., there are 2.5 million burn patients per year, 100,000 of which need hospitalization and 20,000 of which have burns involving more than 20% of the total body surface area (MedPro Month, June 1992).
Many other problems also result from the uncontrolled breakdown of connective tissues by MMPs. These problems include, for example, rheumatoid arthritis; osteoarthritis; osteopenias, such as osteoporosis, periodontitis, gingivitis, corneal epidermal, and gastric ulceration; tumour metastasis, invasion, and growth; neuroinflammatory disorders, including those involving myelin degradation, for example, multiple sclerosis; and angiogenesis dependent diseases, which include angiofibromas, hemangioma, solid tumors, blood-borne tumors, leukemia, metastasis, telangiectasia, psoriasis, scleroderma, pyogenic granuloma, myocardial angiogenesis, plaque neovascularization, coronary collaterals, ischemic limb angiogenesis, corneal diseases, rubeosis, neovascular glaucoma, diabetic retinopathy, retrolental fibroplasia, arthritis, diabetic neovascularization, macular degeneration, wound healing, peptic ulcer, fractures, keloids, vasculogenesis, hematopoiesis, ovulation, menstruation, and placentation.
Given the large number of diseases associated with MMP activity, there is a need to control MMP activity. Several approaches have been suggested to accomplish such regulation. One approach has focused on the catalytic role of zinc in MMPs, and designing zinc chelating regulators. Potent regulators have been generated by introducing zinc chelating groups, such as peptide hydroxamates and thiol-containing peptides, into substrates. Peptide hydroxamates and TIMPs have been successfully used in animal models to treat cancer and inflammation. While these hydroxamates are potent as regulators of MMPs by binding to zinc, they are toxic to humans because they bind to all zinc-containing enzymes. Because many biochemical reactions occurring in the body require zinc, use of the hydroxamates detrimentally effects these other functions and can result in death.
Other known zinc-chelating MMP regulators are peptide derivatives based on naturally occurring amino acids and are analogues of the cleavage site in the collagen molecule (Odake et al. (1994) Biophys. Res. Comm. 199, 1442–46). Some MMP regulators are less peptidic in structure and may more properly be viewed as pseudopeptides or peptide mimetics. Such compounds usually have a functional group capable of binding to the zinc (II) bound in the MMP. Known compounds include those in which the zinc binding group is a hydroxamic acid, carboxylic acid, sulphydryl, or oxygenated phosphorus (for example, phosphinic acid and phosphonamidate, including aminophosphonic acid) groups.
Other approaches include small molecule regulation (Levy et al. (1998) J. Med. Chem. 41, 199–223; Wojtowicz-Pragaet al. (1997) Invest. New Drugs 15, 61–75; Duivenvoorden, et al. (1997) Invasion and Metas. 17, 312–22) and regulation via anti-MMP antibodies (Su et al. (1995) Hybridoma. 14, 383–90).
More specifically, an elastase inhibitor is disclosed in U.S. Pat. No. 5,734,014 to Ishima et al. Elastase secreted by neutrophils causes tissue damage, and in this process, creates an active abundance of oxygen. Elafin isolated from psoriatics has elastase inhibiting activity. However, this naturally occurring elafin is unstable to oxidation. Ishima developed elafin derivatives that are stable to oxidation so that elastase regulation can be more efficient. The oxidation-stable derivative is created by partly modifying the amino acid sequence of natural elafin. The modification can be created by either chemical synthesis or site-directed mutagenesis.
U.S. Pat. No. 5,464,822 to Christophers et al. discloses a polypeptide that possesses inhibitory activity against human leukocyte elastase. The polypeptides possess inhibitory activity that is specific for serine proteases. For example, they possess inhibitory activity against proteases, such as human leukocyte elastase and porcine pancreatic elastase, but do not possess any significant inhibitory activity against trypsin. These polypeptides can be prepared by genetic engineering or obtained from psoriatic scales of human skin.
U.S. Pat. No. 5,698,671 to Stetler-Stevenson et al. discloses a protein defined by the presence of specific cysteine-containing amino acid sequences, isolated from the conditioned media of cultured human tumor cells, that binds with high affinity to MMPs and analogs thereof. The particular inhibitor is made by preparing peptides and proteins having a cysteine residue at the same interval as that of the various tissue inhibitors of metalloproteinase (TIMPs). The peptides must have at least two appropriately spaced cysteines to ensure inhibitory activity by virtue of a disulfide bridge formation. In addition, the invention discloses a method for purifying natural MMP inhibitors by MMP affinity chromatography.
Despite these varied approaches, the current art does not selectively regulate MMP activity. Traditionally, high affinity regulators have been utilized, resulting in complete MMP inhibition. However, shutting off all MMP activity is actually deleterious to the healing process, as some MMP activity is required for tissue remodelling. For example, potent inhibition aimed at binding the zinc (II) site is toxic to humans because it shuts off bind to all zinc-containing enzymes. It is therefore necessary to have regulation be selective.
Thus, there is a need in the art for improved regulation of MMPs to promote healing of chronic and acute wounds.
There is also a need in the art for an inhibitor having relatively good affinity, which is selective.
Furthermore, there is a need in the art for MMP inhibitors that are not toxic to the individual to whom they are administered.